The ability of neurons and their axons to migrate and make precise connections is important during development, learning and memory formation, and repair of damage to the nervous system. However, the cytoplasmic signaling systems that allow migrating neurons to change direction in response to guidance cues in the cellular environment are not well understood. The goal of our laboratory is to understand in detail how one important type of cytoplasmic proteins, the MRL proteins, help to connect guidance signals at the cell surface with cellular migratory responses. In neurons, the MRL proteins are thought to respond to guidance cues by asymetrically localizing cytoplasmic proteins involved in actin polymerization, so that the axon will grow out in a desired direction. However, in T cells, the MRL proteins work instead by activating integrin receptors on the cell surface, so that the T cells is affecting rather than simply responding to the environment (the so-called 'inside out'pathway). We have evidence in the model system C. elegans that the MRL protein MIG-10 may function not only in neurons, but also in a similar 'inside out'pathway in the epidermal cells that provide the substrate on which neurons migrate. Our goal in this proposal is to better understand how MIG-10 functions in both neurons and epidermal cells to direct multiple migrations in C. elegans. In Specific Aim 1, we propose to determine in which cell types each of the three MIG-10 protein isoforms is expressed, and also where their expression is required for function. We will utilize transgenic animals containing genomic constructs that contain all the sequences required for correct expression and function of the isoforms, as well as using cell-specific expression of cDNA constructs to rescue function, and RNAi constructs to knock down function. To better understand the pathway in which the MRL proteins function, we have begun to identify proteins that interact physically with MIG-10 in a yeast two hybrid system. We are focusing on one of these interacting proteins, ABI-1, which is known to be part of the actin polymerization machinery. In Specific Aim 2, we will characterize exactly which migration functions ABI-1 shares with MIG-10, and determine in which cell types ABI-1 is functioning, using a cell-specific rescue/knock down approach similar to that used for MIG-10 in Aim 1. In Specific Aim 3, we will make targeted deletions in both ABI-1 and MIG-10, and determine what protein domains are needed for binding between these two proteins in vitro (using the yeast two hybrid and a cell culture system) and in vivo (by creating transgenes expressing deleted proteins and determining their ability to rescue migrations in the worm.) The studies proposed here will clarify the mechanisms by which MRL proteins work in the nervous system, and in particular how they interact with ABI-1 to regulate actin polymerization. PUBLIC HEALTH RELEVANCE: As the nervous system forms, nerve cells migrate to reach their correct locations and make the specific connections with each other that are vital to correct nervous system function. MIG-10, a protein important to these migrations, was recently shown to be required for nerve cell regeneration following damage, while a closely related protein is in a signaling pathway involved in Alzheimer's Disease. Thus, our study of how the MIG-10 protein functions will provide basic knowledge necessary to better understand and eventually treat these conditions.